The complete chloroplast genome of Primula amethystina subsp. argutidens (Primulaceae)

Abstract Primula amethystina subsp. argutidens (Franchet) W. W. Smith & H. R. Fletcher (1942) is a blooming plant of the family Primulaceae. Here, we sequenced, assembled, and annotated the complete chloroplast (cp) genome of P. amethystina subsp. argutidens. The cp genome of P. amethystina subsp. argutidens is 151,560 bp in length with a GC content of 37%. The assembled genome has a typical quadripartite structure, containing a large single-copy (LSC) region of 83,516 bp, a small single-copy (SSC) region of 17,692 bp, and a pair of inverted repeat (IR) regions of 25,176 bp. The cp genome contains 115 unique genes, including 81 protein-coding genes, four rRNA genes, and 30 tRNA genes. Phylogenetic analysis showed that P. amethystina subsp. argutidens was closely related to P. amethystina.

). In Flora of China, P. amethystina subsp. argutidens is classified as a subspecies of P. amethystina (Hu and Kelso 1996). This subspecies is a perennial herb with rich violet-blue flowers and deeply serrated leaves, mainly distributed in alpine grasslands at an altitude of 3500-5000 m in the western Sichuan Province of China (Hu and Kelso 1996). Compared with P. amethystina and P. amethystina subsp. brevifolia, P. amethystina subsp. argutidens has a more petite stem body, deeper-toothed leaf margins, shorter pedicels, and a profoundly recessed corolla lobe end (Hu and Kelso 1996). Although cp genome is widely used in phylogenetic research and molecular identification in many families of angiosperm, plastome phylogenomic studies of the phylogenetic relationship between P. amethystina subsp. argutidens and P. amethystina are lacking (Huang et al. 2017;Bi et al. 2018). Here, we report the first complete cp genome of Primula amethystina subsp. argutidens to provide a plastome database for further phylogenetic analysis.

Materials and methods
In this study, P. amethystina subsp. argutidens was sampled from Shangri-la of Yunnan Province, China (27 37 0 11 00 N, 99 38 0 29 00 E). Voucher specimens  were deposited in the Herbarium of Yunnan Normal University (YNUB, Figure 1. P. amethystina subsp. argutidens species reference image was taken in Shangri-la, Yunan Province, China (27 37 0 11 00 N, 99 38 0 29 00 E) and provided by corresponding author Huang Yuan. It is a perennial herb with rich violet-blue flowers and deeply serrated leaves.
Website: https://life.ynnu.edu.cn/, Contact: Jian-Lin Hang, Email: hjlynub@163.com). Total genomic DNA was extracted using a modified CTAB method (Porebski et al. 1997). The fragmented genomic DNA was used to construct short-insert libraries for Illumina paired-end (PE) sequencing on the Illumina Hiseq X Ten sequencer. We obtained 17,593,572 filtered reads and then assembled the plastid genome using the software NOVOPlasty v2.7.2 (Dierckxsens et al. 2017) with P. amethystina as the reference genome (GenBank accession no. NC_053577). Then, we used Geneious V2020.1.1 software (Kearse et al. 2012) to annotate the cp genome of P. amethystina subsp. argutidens using P. amethystina (Wang et al. 2021) as a reference and then used Primula bulleyana (Chen, Zhang et al. 2019) as a reference for modification and correction. The annotated cp genome sequences of P. amethystina subsp. argutidens were deposited in the GenBank database under the accession no. ON416902. The circular Primula amethystina subsp. argutidens genomic map and schematic map of the cis and trans splicing genes were drawn using CPGView (Liu et al. 2023, http://www.1kmpg.cn/cpgview/). To verify the accuracy of the assembly, we mapped clean reads to the assembled chloroplast (cp) genomes to assess the depth of coverage ( Figure S1). We used DnaSP6.12 to perform a sliding window analysis of nucleotide diversity to evaluate the divergence of the four Sect. Amethystina cp genome sequences (Librado and Rozas 2009). argutidens chloroplast genome, generated by CPGview (http://www.1kmpg.cn/cpgview/). The species' name is shown in the top left corner. The map contains six tracks by default. From the center outward, the first track shows the dispersed repeats. The dispersed repeats consist of direct (D) and palindromic (P) repeats, connected with red and green arcs. The second track shows the long tandem repeats as short blue bars. The third track shows the short tandem repeats or microsatellite sequences as short bars with different colors. The colors, the type of repeat they represent, and the description of the repeat types are as follows. The small single-copy (SSC), inverted repeat (IRa and IRb), and large single-copy (LSC) regions are shown on the fourth track. The GC content along the genome is plotted on the fifth track. The genes are shown on the sixth track. The optional codon usage bias is displayed in the parenthesis after the gene name. Genes are color-coded by their functional classification. The transcription directions for the inner and outer genes are clockwise and anticlockwise, respectively. The functional classification of the genes is shown in the bottom left corner. In order to further determine the phylogenetic relationship of P. amethystina subsp. argutidens within the genus Primula, the multiple sequence alignment of the complete cp DNA of 47 species from the Primulaceae was analyzed by MAFFT (Katoh and Standley 2013). Four Lysimachia species, four Androsace species, and Glaux maritima were chosen as outgroups for constructing a phylogenetic tree. The maximum-likelihood (ML) tree was constructed using IQ-TREE v1.6.10 (Nguyen et al. 2015), and the best-fit model according to the Bayesian information criterion (BIC) was TVM þ F þ R3 (Kalyaanamoorthy et al. 2017). Branch supports were tested using ultrafast bootstrap (UFBoot) (Hoang et al. 2018) and SH-like approximate likelihood ratio test (SH-aLRT) (Guindon et al. 2010) with 10,000 replicates.

Results
The complete cp genome of P. amethystina subsp. argutidens was 151,560 bp in length with an overall GC content of 37% and an average coverage of 1789.6 ( Figure 2, Figure S1). The assembled genome has a typical quadripartite structure, containing a large single-copy (LSC) region of 83,516 bp and a small single-copy (SSC) region of 17,692 bp, which are separated by a pair of inverted repeat (IR) regions of 25,176 bp. The cp genome contains 134 genes, including 89 proteincoding genes, eight rRNA genes, and 37 tRNA genes, of which 81 CDS genes, four tRNA genes, and 30 rRNA genes are unique, respectively. Totally, 11 cis-splicing genes including rps16, atpF, rpoC1, pafl, clpP, petB, petD, rpl16, rpl2, ndhB, and ndhA ( Figure S2A), and one trans-splicing genes rps12 ( Figure S2B) were detected. The cp genomes of four species of Sect. Amethystina were compared to screen singlenucleotide polymorphisms (SNPs) with higher nucleotide diversity value. As a result, the four most variable regions were rpl2-trnH-GUG, trnH-GUG-psbA, trnK-UUU-rps16, and rpoB-trnC-GCA of which three are located in the LSC region and one is in the SSC region. Partial sequences of trnH-GUG, psbA, rps16, rpoB, and ycf1 also have rich variable sites (Table S1, Figure S3). The phylogenetic tree demonstrated that P. amethystina subsp. argutidens and P. amethystina formed a robust monophyletic clade, which was sister to each other ( Figure 3).

Conclusions
Our result supported well the subspecies of P. amethystina subsp. argutidens. The complete cp genome sequence of P. amethystina subsp. argutidens provided a valuable genomic database for further studies on the evolutionary history and phylogenetic relationship of Primula species.

Ethical approval
This article does not contain any studies with human participants or animals performed by any authors. The species described in this paper are not endangered, protected, or personally owned. The plant material was collected in accordance with guidelines provided by the authors' institution (School of Life Sciences, Yunnan Normal University) and national regulations.

Author contributions
Yuan Huang and Shu-Bao Wang conceived and designed this study. Shu-Bao Wang, Yun-Qi Liu, and Li Zhang conducted an analysis. Shu-Bao Wang and Rui Li contributed to the analytical methods. Shu-Bao Wang wrote the manuscript. Yuan Huang revised the manuscript. All authors have read and agreed to the published version of the manuscript.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Funding
The research was supported by the National Natural Science Foundation of China [No. 31960050]; the Yunnan Education Department Grant [No. 2021Y500]; and the Major Project on Biodiversity Conservation of the Ministry of Ecology and Environment.

Data availability statement
The genome sequence data supporting the study's findings are openly available in NCBI GenBank at https://www.ncbi.nlm.nih.gov/ under accession no. ON416902. The associated BioProject, SRA, and Bio-Sample numbers are PRJNA834988, SRR19175835, and SAMN28088488, respectively.